Yeast strains, such as Pichia pastoris, are commonly used for the production of heterologous proteins. P. pastoris has become a popular model system for the study of peroxisome biogenesis (Gould et al., Yeast 8:613-628 (1992)), autophagy (Tuttle and Dunn, J. Cell Sci. 108:25-35 (1995); Sakai et al., J. Cell Biol. 141:625-636 (1998)) and the organization and biogenesis of the organelles of the secretory pathway (Rossanese et al., J. Cell Biol. 145:69-81 (1999)). The development of simple DNA transformation systems (see Cregg et al., Mol. Cell. Biol. 5:3376-3385 (1985)) and the availability of selectable marker genes have been of great importance in conducting the above experiments. Currently, the biosynthetic marker genes ADE1, ARG4, HIS4 and URA3 are used in conjunction with the corresponding auxotrophic host strains to select for transformed cells. Lin Cereghino et al., Gene 263:159-169 (2001). The use of dominant selectable markers to identify transformants is also possible, but markers are limited to the Sh ble gene from Streptoalloteichus hindustanus, which confers resistance to the drug Zeocin (Higgins et al., Methods Mol. Biol. 103:41-53 (1998)), and the blasticidin S deaminase gene from Aspergillus terreus, which confers resistance to the drug blasticidin (Kimura et al., Mol. Gen. Genet. 242:121-129 (1994)).
Stable integration of cloned DNA segments into the yeast genome through homologous recombination is well known in the art. See e.g., Orr-Weaver et al., Proc. Natl. Acad. Sci. USA 78:6364-6358 (1981). More recently, methods have been developed in S. cerevisiae to generate yeast strains containing DNA integrated at multiple unlinked sites by homologous recombination using molecular constructs containing the URA3 marker gene. See e.g., Alani et al., Genetics 116: 541-545 (1987). In this approach, a construct is generated in which the S. cerevisiae URA3 gene is flanked by direct repeats of a Salmonella hisG DNA. This construct is inserted into a cloned target gene of interest and the linear cassette, containing the complete URA3 gene flanked by direct repeats from hisG and further flanked by 5′ and 3′ segments from the target gene, is introduced into a Ura3− mutant yeast strain by transformation. Stable integrants arising from homologous recombination at the genomic locus of the target gene linked to the URA3 marker gene are then isolated by selection for growth in the absence of uracil. Excision of the URA3 gene through a recombination event between the flanking hisG direct repeat segments restores uracil auxotrophy (Ura−) but leaves behind a disrupted genomic copy of the target gene. Ura+ strains of S. cerevisiae are unable to grow on medium supplemented with the pyrimidine analog 5-fluoroorotic acid (5-FOA) whereas Ura− cells survive such treatment. Cells lacking the URA3 gene are thus readily identified using a positive counterselection on 5-FOA. Boeke et al., Mol. Gen. Genet. 197:345-346 (1984). Through repeated use of the recyclable URA3 marker construct, multiple different genes of interest can be disrupted within a single strain. Similar approaches have been used in other fungi. Wilson et al., Yeast 16:65-70 (2000).
Extensive genetic engineering projects, where several genes in parallel have to be expressed and several others have to be eliminated, require the use of counterselectable markers and plasmids for stable genetic integration of heterologous proteins into the host genome. Recently, a new counterselectable marker based on the T-urfl3 gene from the mitochondrial genome of male-sterile maize has been described for P. pastoris. Soderholm et al., BioTechniques 31:306-312 (2001). Toxicity of the T-urfl3 gene appears to be a host specific problem, however, as the gene may be conditionally lethal with certain gene disruptions that are otherwise not lethal. In addition, the gene is also toxic in the absence of the counterselecting agent methomyl, therefore, the counterselection step must be performed immediately. In addition, a separate gene is required for the initial positive selection step, and the agent used for the counterselection step, methomyl, is light sensitive and breaks down rapidly in aqueous solutions. The system is therefore more complicated than the URA3 system described above, in which the same gene is responsible both for the initial selection of Ura+ prototrophs and for the subsequent counterselection of Ura− auxotrophs. It would be useful to find new marker genes in the yeast pyrimidine biosynthetic pathway in which selection of auxotrophs and counterselection using 5-FOA or similarly acting agents may be used to select and counterselect a single marker gene in multiple rounds of genetic transformation at different loci.
Five structural genes providing six enzymatic steps are responsible for endogenous pyrimidine biosynthesis in S. cerevisiae. Montigny et al., Mol. Gen. Genet. 215:455-462 (1989). The last two steps in this pathway, the conversion of orotic acid to orotidine 5′-phosphate and the conversion of orotidine 5′-phosphate to uridine 5′-phosphate, are catalyzed by orotate phosphoribosyltransferase (OPRTase) and orotidine-5′-phosphate decarboxylase (OMPdecase). These enzymes are encoded by the URA5 gene and the URA3 gene, respectively. Both genes have been cloned, characterized and used for genetic integration in S. cerevisiae, but only the URA3 gene has been cloned in P. pastoris. 
The S. cerevisiae URA5 gene was cloned by complementation of a non-reverting E. coli pyrE mutant that was blocked in orotate-phosphoribosyl transferase activity. Montigny et al., Mol. Gen. Genet. 215:455-462 (1989). Yeast cells lacking this gene displayed a leaky phenotype, however, indicating that, in S. cerevisiae, another protein possesses orotate-phosphoribosyl transferase activity. See Jund and Lacroute, J. Bacteriol. 109:196-202 (1972). The URA5 gene has also been identified in Kluyveromyces lactis. Bai et al., Yeast 15:1393-1398 (1999). The gene order around the URA5 gene has been examined in S. cerevisiae, K. lactis, C. albicans and Y. lipolytica. Sánchez and Domínguez, Yeast 18:807-813 (2001). In all four organisms, the URA5 gene and a gene which functions in the secretory pathway (the SEC65 gene) are arranged adjacent to one another and in the opposite relative orientation.
A selection system based on disrupting the URA3 gene in P. pastoris has recently been disclosed. U.S. Pat. No. 6,051,419. The methods described therein also provide “pop-in” (site-directed integration of the transforming DNA by gene addition) and “pop-out” (recombination between functional and nonfunctional genes resulting in the loss of one of these genes and the URA3 gene) in what is referred to as a “bidirectional selection process.” “Pop-in/pop-out” gene replacement using S. cerevisiae URA3 is a convenient method because, as described above, the selection marker can be recycled. See Boeke et al., Meth. Enzymol. 154:164-175 (1987). P. pastoris ura3 auxotrophs, however, grow slowly. U.S. Pat. No. 6,051,419. In addition, because the sequences responsible for homologous recombination in the “popping out” step are the same as those responsible for the “popping in” step in a single-crossover recombination process, the genetic material inserted by “pop-in” is likely to be lost by “pop-out”. The method is thus more suitable for generating point mutants or gene disruptions than for stably incorporating expressable heterologous genes of interest into the genome.
Currently available auxotrophic strains of P. pastoris suffer the further disadvantage that the respective auxotrophic marker genes have the potential to revert. A high reversion rate decreases the usefulness of auxotrophic strains, because revertant colonies are misidentified as false-positive transformants.
Given the utility of the URA3 selection and counterselection system in S. cerevisiae and the limitations on using these and other current methods in other yeast and fungi, the identification of a URA5 gene in P. pastoris and the development of a system for selecting stable genetic integration events using URA5 would be useful.